Drug delivery to the brain is hampered by the presence of the blood-brain barrier (BBB). The BBB endothelium is very impermeable and allows only those molecules that have combined properties of low molecular weight (<500 Da) and lipophilicity to enter the brain from the bloodstream. Even molecules with such attributes may be effluxed as substrates for p-glycoprotein or similar transporters. Taken together, these BBB properties conspire to restrict BBB passage of greater than 98% of small molecule pharmaceuticals and nearly 100% of all protein and gene therapeutics. However, if antibodies are used to target receptor-mediated transport systems at the BBB, drug molecules and drug carriers can be effectively transcytosed across the BBB endothelium into brain tissue. Such noninvasive delivery from blood to brain is a result of the antibody acting as a surrogate ligand for the endogenous transport systems. Current known antibody-targeted brain delivery systems include the transferrin and insulin receptor systems. These are expressed ubiquitously throughout the body and lead to mis-targeting of expensive pharmaceuticals.
Receptor-ligand recognition and binding frequently depend on pH-induced changes stemming from the combined protonation states of amino acids within the protein. Histidine is considered a key amino acid driving pH sensitivity having a side-chain pKa of 5.5-6.5 in the context of proteins [1]. Evidence suggests that proteins have adapted to function in a range of subcellular pH environments through non-random placement of histidine residues [2]. These phenomena have been exploited in therapeutic protein design to alter intracellular trafficking. For example, interactions with the neonatal Fc-receptor (FcRn), which functions in a pH dependent manner to regulate serum IgG levels [3], have been modified. The Fc region surrounding critical histidine residues of the monoclonal antibody Motavizumab was mutated improving FcRn binding at pH 6.0 without affecting its affinity at pH 7.2, thereby achieving a 4-fold extension in serum half-life [4-6]. In contrast, desiring a reduction in therapeutic IgG serum half-life, a competitive antibody, or “Abdeg”, was created to bind FcRn tightly at both pH 6.0 and pH 7.2, hence occupying FcRn at the expense of therapeutic antibody binding [7]. While these studies describe the modulation of a preexisting pH-dependent system, it is also possible to introduce pH-sensitive binding. As examples, both the anti-IL6R antibody Tocilizumab [8], and the anti-PCSK9 antibody RN316 [9] were engineered to escape target-mediated degradation by introducing histidine residues at select positions in the antibody CDR loops, so as to induce antibody-antigen dissociation at endosomal pH. Engineering pH-sensitive ligand binding has also been employed to increase the potency of non-immunoglobulin scaffolds as in the case of the cytokine GCSF [10], and the iron carrier protein transferrin [11].
The transferrin receptor (TfR) presents a valuable therapeutic target which can be antagonized directly, or exploited indirectly as an intracellular drug delivery vector. These opportunities result from the ubiquitous expression of TfR on normal cells and elevated expression on cancer cells, as well as the endocytotic route used to transport iron-bearing transferrin inside the cell (reviewed in [12,13]). The natural ligand for TfR, the serum protein transferrin (Tf), circulates in iron-free (apoTf) or iron-bound (holoTf) forms [14,15]. HoloTf binds the transferrin receptor (TfR) tightly at blood pH (7.2-7.4), and the complex is internalized via clathrin-mediated endocytosis (CME) [16]. As holoTf-TfR complexes cycle though acidic endosomes (pH 5.0-6.0), an intricately coordinated series of pH-induced conformational changes induces the release of both iron molecules to yield apoTf, which has an increased affinity for TfR at endosomal pH [15,17-19]. This is followed by recycling of the apoTf-TfR complex to the cell surface (pH 7.2-7.4) where apoTf has a decreased affinity for TfR and dissociates back into the blood stream [17,20]. Cytotoxins based on conjugates of transferrin have been widely studied as therapeutic agents [21]. A detailed kinetic model of the TfR cycle was created and analyzed for routes that might lead to a greater overall cellular association of Tf or Tf conjugates [11]. It was posited that inhibition of iron release from Tf could lead to endosomal dissociation of holoTf that, unlike apoTf, could rapidly rebind at blood pH and participate in further cycles of endocytosis at blood pH [11,17]. Indeed, when Tf was genetically altered to inhibit iron release, diphtheria toxin conjugates of the mutant Tf showed increased cytotoxicity compared to wild-type Tf conjugates [22]. Similarly, it has been shown that improved cytotoxin efficacy for Tf conjugates as well as anti-TfR antibodies is a direct result of increased cellular association [23-25].
Needed in the art is an antibody with pH-sensitive binding capability. Specifically, needed in the art are anti-TfR antibodies that bind TfR in a pH-dependent manner. For example, the needed antibodies could bind TfR at physiological pH and could release TfR rapidly at endosomal pH.